JPH1118769A - Method and system for preparing dna and rna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a preparation method and a system for DNA or RNA enabling the automatic high speed preparations of samples of plasmid DNA, microorganisms and higher organisms, nucleus DNA, etc. SOLUTION: In a preparation method in which DNA or RNA is prepared via plural processes, the time for one step is set at the minimum effective time in the plural processes, and processes needing longer times are divided so that they are carried out in plural steps, and the samples to be processed are moved for every step via plural processes. This system enables the supply and recovery of sample plates at the minimum effective time, and the extreme increase of a processing capacity per unit time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and a system for automatically preparing plasmid DNA and microorganisms, nuclear DNA or RNA of higher organisms, etc. at a high speed.

[0002]

2. Description of the Related Art DNA and RNA represented by plasmids
Is to enable a large amount of protein to be obtained by utilizing a bacterial protein synthesis field by incorporating a specific gene.
It is needed in large quantities in sequencing. Conventionally, there are various methods for preparing a plasmid having such a use, but according to a typical conventional method, a plasmid is introduced into a microbial cell such as Escherichia coli, and the microbial cell is cultured. The plasmid is extracted from the microbial cells multiplied by the above. In order to extract the plasmid from the microbial cells, a lysing solution is added to a culture solution in which the microbial cells have been cultured, the cells are lysed, and the plasmid eluted into the solution by the lysis is separated from the solution and collected.

[0003] As a conventional plasmid DNA preparation apparatus for carrying out such a preparation method, an apparatus which performs purification by a reduced-pressure double filtration method has been developed and is commercially available. In this conventional apparatus, a cell collection step, a lysis step, an alkaline SDS treatment step, a neutralization slow protein treatment step, a vacuum filtration step, a DNA adsorption step, a washing step and a D step
The NA elution step is performed sequentially in a single apparatus in a batch manner.

[0004]

In such a conventional batch-type apparatus, each of the above-described steps must be performed sequentially in one apparatus. It was not possible to proceed to the next processing step only after completion. Therefore, the overall processing speed is greatly affected by the processing step requiring the longest time among the above-described processing steps.

Therefore, in a conventional batch-type plasmid DNA or RNA purification apparatus, it took about 2 hours to process, for example, 48 samples. 1
If it is operated for about 17 hours a day, only about 400 samples can be processed by one apparatus. In order to increase the daily throughput, it is conceivable to extend the daily operating time, but this has a limit. On the other hand, it is conceivable to increase the number of used devices. However, not only does the equipment cost increase, but also the amount of work performed on the devices increases by the number of devices used, resulting in higher costs.

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems of the prior art, and to automatically prepare plasmid D at a high speed.
It is an object of the present invention to provide a method and system for preparing DNA or RNA, which enables preparation of samples such as NA and microorganisms, higher organisms and nuclear DNA.

[0007]

According to one feature of the present invention, in a DNA or RNA preparation method for preparing DNA or RNA through a plurality of steps, one or more of the plurality of processing steps has an effective minimum time. Set the time in the step, the processing step that takes longer time among the plurality of processing steps, the time is divided, performed in a plurality of steps,
The sample to be processed is moved step by step through the plurality of processing steps.

According to another aspect of the present invention, a DNA or RNA prepared from DNA or RNA through multiple processing steps.
In the NA preparation system, a plurality of processing stations arranged in order to perform each of the plurality of processing steps, and a sample plate to be processed are moved step by step through the processing stations sequentially. Sample plate moving means, wherein the processing time in the one step is set to the effective minimum time of the plurality of processing steps, and the processing step requiring a longer time among the plurality of processing steps Is provided in a divided manner so as to perform the processing in a plurality of steps.

According to one embodiment of the present invention, the plurality of processing steps include a cell lysis step, a DNA adsorption step, a solution suction filtration step, a washing step, and a DNA recovery step, and the processing time of one step is reduced. , The effective minimum time of the plurality of processing steps is set.

According to one embodiment of the present invention, the sample plate can perform all of the plurality of processing steps on the same plate. The sample plate of this embodiment comprises a flat plate having a plurality of holes for holding a sample to be processed in a matrix, and a suction port is formed on the bottom side of each of the holes. Is provided with a carrier and a structure for holding the carrier in front of the suction port. Carriers include glass filters, glass beads,
It can be selected from glass powder, silicon beads, ion exchange resin, hydroxyapatite, and celite.

According to another embodiment of the present invention, the DNA or RNA preparation system further comprises an automatic sample supply means for automatically supplying a sample plate to a first one of the plurality of processing stations. Automatic sample storage means for collecting and storing a sample plate from the last processing station of the plurality of processing stations.

[0012]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Next, the present invention will be described in more detail with reference to the accompanying drawings.

FIG. 1 is a block diagram showing the outline of the configuration of an embodiment of the DNA preparation system according to the present invention. As shown in the block diagram of FIG.
The A preparation system mainly includes an automatic sample supply device 100 for automatically supplying a sample plate such as a titer plate carrying a sample such as a culture solution containing microbial cells to be processed to a preparation device; Supply device 100
And a sample storage device for automatically collecting and storing a sample plate from the preparation device 200 after each processing step is completed, by receiving a sample plate automatically supplied from the apparatus and performing each processing step. 300.

The preparation device 200 is used for adsorbing plasmid DNA dissolved in cells or nuclear DNA of microorganisms and higher organisms onto a glass carrier, and adsorbing the plasmid DNA adsorbed on the glass carrier.
Is automatically collected. The preparation apparatus 200 mainly includes a treatment station 210 for performing a plasmid insertion step for receiving a sample carrier, and a cell lysis solution and a DNA adsorption solution sequentially added to microbial cells in the presence of a glass carrier, and the state is allowed to stand. To promote the reaction and adsorb plasmid DNA or microorganisms, and nuclear DNA of higher organisms to the glass carrier. Treatment stations 220 and 230 for the cell lysis step and DNA adsorption step, and suction filtration to separate the added solution from the glass carrier. A processing station 240 for performing a solution suction filtration step to perform the washing, a processing station 250 for performing a washing step for washing the residual solution after the solution suction filtration,
A processing station 260 for performing a DNA recovery step of eluting and recovering with an NA elution solution.

FIG. 2 is a schematic perspective view of a microtiter plate as an example of a sample plate used in the DNA preparation system. As shown in FIG. 2, the microtiter plate 10 has 96 holes (wells) 11 formed in 12 rows × 8 columns in a flat plate. FIG. 3 shows the cross-sectional shape of one of these holes 11. As is well shown in FIG.
A suction port 12 is formed on the bottom side of the hole 11, and a glass filter 13 and a membrane filter 14 are provided on the bottom of the hole 11 in front of the suction port 12 so as to overlap each other. This microtiter plate 10
Is supplied automatically to the preparation device 200 by the automatic sample supply device 100 in a state where the culture solution 1 containing the microbial cells as the sample to be processed is put into each hole 11 as described later. Each processing station 210 by the apparatus 200
After receiving all of the predetermined processing while moving from the preparation apparatus 200 to the processing section 260, the preparation apparatus 200 sends the preparation processing to the automatic sample storage apparatus 300 and collects and stores the same.

Each of the microtiter plates 10 is preferably provided with an identification code such as a bar code for identifying each of them. By doing so, it is possible to recognize a titer plate containing an abnormal state and abnormal bacteria (poor suction), store the data in a recording device, and display the data at any time on a monitor screen. It becomes possible.

FIG. 4 is a schematic front view showing the preparation apparatus 200, and FIG. 5 is a schematic plan view thereof. As shown in FIG. 5, the preparation apparatus 200 includes a plate transport unit 204 (this is a moving line mechanism for automatically moving the titer plate 10 in a substantially U shape along the upper surface of the main body housing). The moving line mechanism itself may be similar to a well-known automatic industrial line mechanism, and will not be described in detail here. The processing stations 210, 220, 230, 240 and 250 as described above are arranged along a U-shaped moving line on the upper surface of the main body housing. Can be installed before or after.

As is well shown in FIG. 5, in the preparation apparatus 200 of this embodiment, the processing station 210 for performing the sample insertion step comprises only the station (place) 1;
The processing stations 220 and 230 for performing the cell lysis step and the DNA adsorption step include three stations 2, 3
And 4 are divided. Further, the processing station 240 for performing the solution suction filtration step includes two stations 5
And 6 are divided. Processing station 250
Are six stations 7, 8, 9, 1 for performing the cleaning process.
8 stations 13, 14, 15, 1 for cleaning and drying
6, 17, 18, 19 and 20. Further, as the processing station 260 for performing the DNA recovery step, the station 21 for performing the TE buffer solution injection step can be provided.

As shown in FIGS. 4 and 5, above the main body housing of the preparation device 200, at a position corresponding to the station 2, a solution 1 is injected into each hole 11 of the microtiter plate 10. A 96-channel precision dispenser 201A for disposing the solution 2 into each hole 11 of the microtiter plate 10 is disposed at a position corresponding to the station 3 at a position corresponding to the station 3. At a position corresponding to the station 4, a 96-channel precision dispenser 201C for injecting the solution 3 into each hole 11 of the microtiter plate 10 is arranged.

At a position corresponding to the station 6, a residual liquid detector 203 is disposed. Furthermore,
At positions corresponding to stations 7, 9 and 11, 96-channel ethanol injectors 202A, 202B and 202C for injecting 80% ethanol solution respectively.
Is arranged. At a position corresponding to the station 13, a 96-channel precision dispenser 201D for injecting an 80% ethanol + 20% glycerol solution is arranged. In the position corresponding to the station 21, TE
A dispenser for injecting a buffer is provided, but in the device of this embodiment, since the DNA is collected at a later stage of the automatic sample storage device, the injector for the TE buffer is not shown. Absent.

Further, in the preparation apparatus 200, a plate separating mechanism 207 is arranged on the upper side of the main body housing on the entrance side of the plate transport section 204, and on the exit side of the plate transport section 204, a plate stacking mechanism 208 is provided. Is arranged. Also, on the front side of the main body housing,
An operation panel 205 and a pressure reduction setting display panel 206 are provided. A suction tank 209 and a tank 211 of an 80% ethanol liquid are arranged on the lower inner side of the main body housing. On the upper part of the main body housing are a tank 212 for solution 1, a tank 213 for solution 2, a tank 214 for solution 3, 80% ethanol + 20
A tank 215 for a% glycerol liquid is arranged.

Here, the solution used in this embodiment will be described. Solution 1 contains 50 mM glucose, 2
5 mM Tris-HCl buffer (pH 8.0), 10 mM
EDTA (pH 8.0), solution 2 was 0.2 N sodium hydroxide, 1% SDS, and solution 3 was 0.7
Potassium acetate (pH 4.8), 5.3 Guanidine hydrochloride. The TE buffer is 10m Tris-HCl buffer (pH 8.0), 1mM EDTA.

FIG. 6 is a schematic plan view of an automatic stacker device used as the automatic sample supply device 100 or the automatic sample storage device 300, and FIG. 7 is a schematic front view thereof. The automatic stacker device 400 is self-supporting and can be moved by a caster with a fixing device, and includes a plate storage delivery unit 401 and a plate transport storage unit 402.
, A transparent cover 403, a plate transport plate 404, a plate supply / delivery unit 405, a connector 406, and a coupling unit 407 for connecting to the preparation device 200.

When this automatic stacker is used as a supply stacker (automatic sample supply) 100, it is a device for storing a sample (titer plate) to be supplied to the preparation device 200. In this embodiment, one unit is used. 2
It has a capacity to accommodate 16 titer plates 10, and can introduce and supply a plate set (12 sheets) to the preparation device 200 (supplied to the plate separation mechanism 207) in accordance with an instruction from the main sequencer. It is operated in coordination with the operation, and is detachable from the preparation device 200.

When the automatic stacker device 400 is used as a discharge stacker device (automatic sample storage device) 300, the sample (titer plate) processed by the preparation device 200 is sequentially carried out and stored.
More than one sample can be stored, and the sample stacked from the preparation device 200 can be received (received from the plate stacking mechanism 208) by the instruction from the main sequencer, and stored in the stacker by the instruction from the main sequencer. It is operated in coordination with the operation of the apparatus 200 and is detachable from the preparation apparatus 200.

Next, the processing operation of the preparation apparatus 200 will be described with reference to a schematic plan view of the preparation apparatus 200 of FIG. 5 and a time chart of the DNA preparation step of FIG. As described above, the preparation apparatus 200 has a line 204 including 21 stations (places) 1 to 21 in this embodiment, and each station performs the following processing.

Station (location) processing 1 Sample titer plate receipt 2 Solution 1 Injection 3 Solution 2 Injection 4 Solution 3-GuHCl injection 5 Suction / filtration 6 Suction / filtration, liquid level detection in well 7 Injection of 80% ethanol + Suction / filtration 8 Suction / filtration 9 80% ethanol injection + suction / filtration 10 Suction / filtration 11 80% ethanol injection + suction / filtration 12 Suction / filtration 13 80% ethanol + 20% glycerol injection 14-20 suction / filtration 21 Empty (TE buffer solution) May be injection)

In this embodiment, the time T (minutes) in the horizontal column and the location (station) in the vertical column of the time chart of FIG.
As indicated by the numbers, the time during which line 204 is stopped at each station is typically 2.5 minutes, whereas
It is possible to adjust the time from 0.2 to 3 times.

In the above description of the embodiment, the required time at each station is set to 2.5 minutes.
The present invention is not limited to this, and can be set to an arbitrary time in consideration of an effective minimum time among processing times required for each of a plurality of necessary processing steps.

[0030]

According to the present invention, since the processing operation is divided into several steps in the long-time processing step and the sample plate is moved through these steps according to the contents of each processing step, the sample plate moving type is adopted. In addition, the sample plate can be supplied to the apparatus and collected in an effective minimum time, and the processing capacity per unit time can be significantly increased.

Dispensing an automatic stacker for automatically supplying and recovering a sample plate to a preparation device for performing various processes on the sample plate allows dispensing of the sample to the sample plate, etc. Can be prepared in another place, so that the work of the operator can be performed more efficiently.

[Brief description of the drawings]

FIG. 1 is a block diagram showing an outline of a configuration of an embodiment of a DNA preparation system according to the present invention.

FIG. 2 is a schematic perspective view of a microtiter plate as an example of a sample plate used in a DNA preparation system.

FIG. 3 is a diagram showing a cross-sectional shape of one of the holes of the sample plate of FIG. 2;

FIG. 4 is a schematic front view showing a preparation apparatus as one embodiment of the present invention.

FIG. 5 is a schematic plan view of the preparation device of FIG.

FIG. 6 is a schematic plan view of an automatic stacker device used as an automatic sample supply device or an automatic sample storage device.

FIG. 7 is a schematic front view of the automatic stacker of FIG. 6;

FIG. 8 is a diagram showing a time chart of a DNA preparation step in a DNA preparation system as one example of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Culture solution 10 Microtiter plate 11 hole 12 Suction port 13 Glass filter 14 Membrane filter 100 Automatic sample supply device 200 Preparation device 201A Precision dispenser 201B Precision dispenser 201C Precision dispenser 201D Precision dispenser 202A Ethanol dispenser Container 202B Ethanol dispenser 202C Ethanol dispenser 203 Residual liquid detector 204 Plate transport unit 205 Operation panel 206 Depressurization setting display panel 207 Plate separation mechanism 208 Plate stacking mechanism 209 Suction tank 211 Ethanol tank 212 Solution tank 213 Solution tank 214 Solution Tank 215 Solution tank 210 Sample insertion process processing station 220 Lysis process processing station 230 DNA adsorption process processing station 240 Solution suction filtration process processing Station 250 washing process processing station 260 DNA recovery process processing station 400 automatic stacker

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Katsumi Senoo 2-1 Hirosawa, Wako-shi, Saitama Pref., RIKEN (72) Inventor Tokuji Tachiuchi 2-1 Hirosawa, Wako-shi, Saitama Pref., RIKEN (72) Inventor Shigenari Hisagizaki 1001 Arahata, Tokorozawa-shi, Saitama Lifetech Co., Ltd.

Claims (6)

[Claims] 1. A DNA or RNA preparation method for preparing DNA or RNA through a plurality of steps, wherein a time of one step is set as an effective minimum time among the plurality of processing steps, Among the processing steps that require a longer time, the processing time is divided, and the processing is performed in a plurality of steps.
A DNA or RNA preparation method characterized in that the DNA or RNA is moved step by step. 2. The method according to claim 1, wherein the plurality of processing steps include a cell lysis step,
2. The DNA or RNA preparation according to claim 1, comprising a DNA or RNA adsorption step, a solution suction filtration step, a washing step, and a DNA or RNA recovery step, wherein the processing time of one step is set to an effective minimum time of the plurality of processing steps. Method. 3. A DNA or RNA preparation system for preparing DNA or RNA through a plurality of processing steps, comprising: a plurality of processing stations sequentially arranged for performing each of the plurality of processing steps; Sample plate moving means for sequentially moving the sample plate to be processed for each step, wherein the processing time in said one step is set to the effective minimum time of said plurality of processing steps And a processing station for performing a processing step requiring a longer time among the plurality of processing steps is provided so as to be divided into a plurality of steps so as to perform the processing. Preparation system. 4. The DNA or RNA preparation system according to claim 3, wherein the sample plate can perform all of the plurality of processing steps on the same plate. 5. The sample plate comprises a flat plate having a plurality of holes for holding a sample to be processed in a matrix, and a suction port is formed on a bottom side of each of the holes. The D according to claim 4, wherein a carrier and a structure for holding the carrier are provided in front of the suction port at the bottom.
NA or RNA preparation system. 6. An automatic sample supply means for automatically supplying a sample plate to a first processing station of the plurality of processing stations, and collecting a sample plate from a last processing station of the plurality of processing stations. 6. The DNA or RN according to claim 3, further comprising an automatic sample storage means for storing the sample or sample.
A preparation system.